Hach Company TNTplus 835/836 Nitrate Method 10206

Spectrophotometric Measurement of Nitrate in Water

and Wastewater
 Hach Company TNTplus 835/836 Method 10206
Revision 2.1
January 10, 2013

Spectrophotometric Measurement of Nitrate in Water and Wastewater

1.0 Scope and Application

1.1 These procedures cover the determination of nitrate in drinking water, surface water,
domestic and industrial wastes.

1.2 The method is applicable in the range from 0.20 to 35.0 mg/L NO3- N.

1.3 This method is equivalent or better in performance to SM 4500-NO3- E, EPA 353.2, and
EPA 300.0 for the purposes of regulatory reporting of nitrate and nitrate-nitrite.

2.0 Summary of Method

2.1 The Hach TNTplus Nitrate chemistry follows classical electrophillic aromatic
substitution in that nitrate in the presence of sulfuric acid yields a nitronium ion (+NO2)
and HSO4-. Nitronium ions are electrophiles that attack the aromatic ring of the
dimethylphenol reagent to form intermediate nitro-carbonium ions. The basic HSO4- ion
then extracts a hydrogen ion from the nitro-carbonium intermediate to yield a stable
substitution product (o, or p-nitro-dimethylphenol). The nitrodimethylphenol product is a
highly colored (directly related to the nitro functional group), quantifiable by its visible
absorption spectra. Test results are measured at 345 nm.

3.0 Interferences
3.1 The items listed in the Interfering substances table have been individually checked up to
the given concentrations and do not cause interference. The cumulative effects and
influence of other ions have not been determined. High loads of oxidizable organic
substances cause the reagent to change color and to give high-bias results. The test can
thus only be used for wastewater analyses if the chemical oxygen demand (COD) is less
than 500 mg/L. Measurement results can be verified using sample dilutions or standard
additions.

3.2 Nitrite concentrations of more than 2.0 mg/L interfere (high-bias results). Add 50 mg of
sulfamic acid (amidosulfonic acid) to 5.0 mL of sample, dissolve and wait for 10
minutes. Analyze the prepared sample as described in the procedure above.

Interfering Interference level Interfering Interference level

substance (mg/L) substance (mg/L)
Ag+ 100 Cu2+ 50
Cl- 500 Ca2+ 50
Fe3+ 50 NO2- 2
K+ 500 Cd2+ 50
Na+ 500 Sn2+ 50
Ni2+ 50 Cr6+ 5
Pb2+ 50 Fe2+ 10
Zn2+ 50 Co2+ 10

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3.3 Residual chlorine does not cause an interference with this method.

4.0 Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
determined; however, each chemical should be treated as a potential health hazard.
Exposure to these chemicals should be reduced to the lowest possible level. It is
suggested that the laboratory perform personal hygiene monitoring of each analyst using
this method and that the results of this monitoring be made available to the analyst.

4.2 Unknown samples may contain high concentrations of volatile toxic compounds. Sample
containers should be opened in a hood and handled with gloves to prevent exposure.

4.3 This method does not address all safety issues associated with its use. The laboratory is
responsible for maintaining a safe work environment and a current awareness file of
OSHA regulations regarding the safe handling of any chemicals specified in this method.
A reference file of material safety data sheets (MSDSs) should be available to all
personnel involved in these analyses. Additional information on laboratory safety can be
found in Sections 16.3 and 16.4.

5.0 Equipment

Note: Brand names, suppliers, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance may be achieved using apparatus and
materials other than those specified here, but demonstration of equivalent performance
that meets the requirements of this method is the responsibility of the laboratory.

5.1 Sampling equipment

5.1.1 Sample collection bottles Preferably use polyethylene bottles for collecting and storing
samples for nitrate analysis. Glass bottles are satisfactory if previously they have not
contained high-nitrate solutions.

5.1.2 Cleaning

5.1.2.1 All glassware used should be washed with hot 1:1 HCl and rinsed with distilled water.
Preferably, this glassware should be used only for the determination of nitrate and after
use it should be rinsed with distilled water and kept covered until needed again. If this is
done, the treatment with 1:1 HCl is only occasionally required.

6.0 Equipment for sample analysis

6.1 Hach Company DR 5000, DR 3800, or DR 2800 spectrophotometer

6.2 Equipment for standard preparation

6.2.1 Volumetric flask Glass, 1000-mL.

6.2.2 Volumetric pipette Glass, assorted sizes.

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7.0 Reagents and Standards
7.1 Reagent water Water in which nitrate is not detected at or above the method level of
this method. Bottled distilled water, or water prepared by passage of tap water through
ion exchange and activated carbon have been shown to be acceptable sources of reagent
water.

7.2 Hach Company TNTplus Nitrate Reagent, Cat. No. TNT835 or TNT836.

7.3 Hach Company Nitrate Standard Solutions: 100 mg/L as NO3--N (Cat. No. 194749) and
1000 mg/L as NO3--N (Cat. No.1279249).

7.4 Method detection limit (MDL) solution

7.4.1 Prepare 7 or more replicate MDL solutions by diluting 3.0 mL of the 100 mg/L standard
spiking solution (Section 7.3) to 1000 mL. Final concentration = 0.3 mg NO3--N /L.

7.5 Initial precision and recovery (IPR) solution

7.5.1 Prepare 4 or more replicate IPR solutions by diluting 5.0 mL of the 1000 mg/L standard
spiking solution (Section 7.3) to 1000 mL. Final concentration = 5 mg NO3--N /L.

8.0 Sample Collection, Preservation and Storage

8.1 Samples may be collected in clean glass or plastic bottles.

8.2 Analyze samples as soon as possible. If immediate analysis is not possible, store at 6
C. Samples to be used for measurement of nitrate only must not be preserved with acid
and must be and analyzed within 48 hours of collection.

8.3 If longer storage is required (up to 28 days), adjust sample pH to less than 2 with sulfuric
acid (about 2 mL per liter). Sample refrigeration is still required. If sample is acid
preserved, results must be reported as nitrate-nitrite, also known as total nitrate.

9.0 Quality Control

9.1 It is recommended that each laboratory that uses this method be required to operate a
formal quality assurance program (16.1). The minimum requirements of this program
consist of an initial demonstration of laboratory capability and ongoing analyses of
laboratory prepared water standards as a test of continued performance to assess accuracy
and precision. Laboratory performance is compared to established performance criteria
to determine if the results of analyses meet the performance characteristics of the method.

9.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable
accuracy and precision with this method. This ability is established as described in
Section 9.2. The laboratory shall, on an ongoing basis, demonstrate through analysis of
the ongoing precision and recovery sample that the analysis system is in control.

9.1.2 Accompanying QC for the determination of nitrate is required per analytical batch. An
analytical batch is a set of samples processed during a contiguous 8-hour period. Each
analytical batch must be accompanied by a laboratory reagent blank (LRB), an ongoing
precision and recovery sample (OPR), a matrix spike sample (MS), and a matrix spike

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 duplicate sample (MSD) resulting in a minimum of five analyses (1 LRB, 1 OPR, 1
sample, MS, and MSD).

9.2 Initial demonstration of laboratory capability.

9.2.1 To establish the ability to detect nitrate the analyst shall determine the MDL per the
procedure in 40 CFR 136, Appendix B (16.2) using the apparatus, reagents, and standards
that will be used in the practice of this method. The analyst also shall calculate the
Minimum Level (ML) of quantitation by multiplying the MDL by 3.18 and rounding to
the number nearest to (1,2 or 5) x 10n, where n is a positive or negative integer. The
calculated MDL should be less than or equal to the MDL in Section 13.0 prior to the
practice of this method. Similarly, the calculated ML should be less than or equal to the
ML in Section 13.0

9.2.2 Prepare and measure seven replicates of the MDL standard according to the procedure
beginning in Section 7.4.1.

9.3 Initial precision and recovery (IPR) - To establish the ability to generate acceptable
precision and accuracy, the analyst shall perform the following operations:

9.3.1 Prepare and measure four samples of the IPR standard according to the procedure
beginning in Section 7.5.

9.3.2 Using the results of the set of four analyses, compute the average percent recovery (x)
and the standard deviation of the percent recovery (s) for nitrate. Use the following
equation for calculation of the standard deviation of the percent recovery:

x 2

x n
2 i
i
s
n 1

where:

n = Number of samples
xi = % recovery in sample i

9.3.2.1 Using the values of x and s calculated in the previous step, calculate the Relative
Standard Deviation (RSD) using the equation below:
s
RSD * 100%
X

9.3.2.2 Compare RSD and x with the corresponding limits for initial precision and recovery in
Table 1. If RSD and x meet the acceptance criteria, system performance is acceptable and
analysis of samples may begin. If, however, RSD exceeds the precision limit or x falls

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 outside the range for recovery, system performance is unacceptable. In this event correct
the problem, and repeat the test.

9.4 Laboratory Reagent Blank (LRB) The laboratory reagent blank (LRB) is an aliquot of
reagent water that is treated exactly as a sample including exposure to all glassware,
equipment and reagents that are used with other samples, The laboratory must analyze at
least one LRB with each batch of samples. Data produced are to asses contamination
from the laboratory environment. Values that exceed the MDL indicate laboratory or
reagent contamination should be suspected and corrective action should be taken before
continuing the analysis.

9.5 Ongoing precision and recovery (OPR) - To demonstrate that the analysis system is in
control, and acceptable precision and accuracy is being maintained with each analytical
batch. An analytical batch is a set of samples processed during a contiguous 8-hour
period, not to exceed 20 samples. The analyst shall perform the following operations:

9.5.1 Prepare a precision and recovery standard with each analytical batch.

9.5.1.1 At the end of each analytical batch of samples, analyze a precision and recovery standard.
If the recovery is within the acceptable range of 90 -110%, measurement process is in
control and analysis of samples may proceed. If, however, the recovery is not in the
acceptable range, the analytical process is not in control. In this event, correct the
problem, re-analyze analytical batch, repeating the ongoing precision and recovery test.

9.5.1.2 The laboratory should add results that pass to IPR and previous OPR data and update QC
charts to form a graphic representation of continued laboratory performance. The
laboratory should also develop a statement of laboratory data quality for each analyte by
calculating the average percent recovery (R) and the standard deviation of the percent
recovery (sr). Express the accuracy as a recovery interval from R - 2sr to R + 2sr. For
example, if R = 95% and sr = 5%, the accuracy is 85% to 105%.

9.5.1.3 Depending upon specific program requirements, field replicate spikes may be required to
assess the precision and accuracy of the sampling and sample transporting techniques.

9.6 Matrix Spike and Matrix Spike Duplicate Precision and Recovery (MS/MSD) The
laboratory must, on an ongoing basis, spike at least 5% of the samples from each
analytical batch as defined in Section 9.4.

9.6.1. The concentration of the spike in the sample should be determined as follows:

9.6.1.1 If, as in compliance monitoring, the concentration of nitrate in the sample is being
checked against a regulatory concentration limit, the spike should be at that limit or one
to five times higher than the background concentration determined in Section 11,
whichever concentration would be larger.

9.6.1.2 If the concentration of nitrate in the sample is not being checked against a a regulatory
concentration limit, the spike should be at 5 mg/L or one to five times higher than the
background concentration determined in Section 10, whichever concentration would be
larger.

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9.6.2 Analyze one sample aliquot to determine the background concentration (B).

9.6.3 Analyze the spiked MS and MSD aliquots following the procedure in Section 11.

9.6.5 Calculate each percent recovery (P) as 100 (A-B)%/T, where A is the concentration of
TKN in the spiked samples and T is the known true value of the spike.

9.6.5.1 Compare the percent recovery (P) TKN with the corresponding QC acceptance criteria
found in Section 17, Table 3.

9.6.6 Calculate the relative percent difference (RPD) between two sample results using the
following equation:
D1 D2
RPD x 100
( D1 D 2) / 2
Where, D1 = Concentration of analyte in the MS, D2 = Concentration of analyte in
the MSD.

9.6.6.1 Compare the calculated RPD with the corresponding QC acceptance criteria found in
Section 17, Table 3.

10.0 Calibration and Standardization

10.1 The Hach DR series spectrophotometers have a built-in calibration that is automatically
used when the TNTplus nitrate vial is inserted into the instrument. No further initial
calibration is required. However, the instruments have the capability of developing a
user-calibration. See manufacturers manual for instructions.

10.2 Calibration Verification

10.2.1 To verify that the instrument is measuring nitrate properly, analyze a 0.3 mg/L and 10.0
mg/L nitrate nitrogen standard. Results should be within 10 percent of the actual value.
Perform this calibration verification daily while instrument is in use.

11.0 Procedure
11.1 Instrument Setup follow the instrument manufacturers instructions for instrument
setup.

11.2 Preparation

11.2.1 For LR TNT 835: Pipet 1.0 mL of sample into the reagent vial.
For HR TNT836: Pipet 0.2 mL of sample into the reagent vial.

11.3 Reaction

11.3.1 For LR TNT835: Pipet 0.2 mL of Solution A into the vial.

For HR TNT836: Pipe 1.0 mL of Solution A into the vial.

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11.3.2 Cap and invert the reaction tube 2-3 times until no more streaks can be seen in the
reaction tube solution.

11.3.3 React for 15 minutes.

11.4 Analysis

11.4.1 Wipe the vial and insert the prepared vial into the spectrophotometer. The instrument
reads the barcode, then selects and performs the correct test. No zero is required. Results
are in mg/L NO3--N

12.0 Data Analysis and Calculations

12.1 Nitrate concentration is calculated automatically against internal instrument calibration.

13.0 Method Performance

Performance of the method was demonstrated in multi-lab studies comparing the method
against currently promulgated nitrate methods. The method was evaluated in low ionic
strength (LIS) and high ionic strength (HIS) matrices as well as multiple geographically
diverse finished drinking water samples obtained from both surface water and ground
water sources.

Acceptance Criterion Section Limit

Method Detection Limit 9.2.1 0.05 mg/L NO3- N

Method Limit 9.2.1 0.20 mg/L NO3- N

Initial Recovery Range 9.3.1 90% - 110%

Initial Precision (RPD) 9.3.1 10%

Matrix Recovery Range 9.5.1 90- 110%

14.0 Pollution Prevention

14.1 Follow guidelines in Section 15.

15.0 Waste Management

15.1 It is the laboratorys responsibility to comply with all federal, state, and local regulations
governing waste management, particularly the hazardous waste identification rules and
land disposal restrictions, and to protect air, water, and land by minimizing and control
all releases from fume hoods and bench operations. Compliance with all sewage
discharge permits and regulations is also required.

15.2 For further information on waste management, consult The Waste Management manual
for Laboratory Personnel, and Less is Better: Laboratory Chemical Management for
Waste Reduction, both available from the American Societys Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C.
20036.

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16.0 References
16.1 Handbook of Analytical Quality Control in Water and Wastewater Laboratories,
USEPA, EMSL-CI, Cincinnati, OH 45268, EPA-600-4-79-019, March 1979.

16.2 40 CFR 136, Appendix B.

16.3 OSHA Safety and Health Standards, General Industry, (29 CFR 1910), Occupational
Safety and Health Administration, OSHA 2206 (Revised, January 1976)

16.4 Safety in Academic Chemistry Laboratories, American Chemical Society, Committee

on Chemical Safety, 3rd Edition, 1979.

16.5 Water Analysis Handbook, Hach Company, 5th Edition, 2008.

17.0 Tables
17.1 Acceptance Criteria for Performance tests The QC performance criteria for this method
was performed with a Hach Company DR2800 Spectrophotometer and TNTplus 835
Reagent.

Table 1. Initial Precision and Recovery Method Performance

IPR Spike Lower Limit of Recovery Upper Limit of Recovery

Maximum RSD (%)
Concentration (%) (%)

5.0 mg/L NO3- N 10 90 110

Table 2. Method Detection Limit (MDL) & Minimum Level (ML) of Quantitation

MDL Test
Concentration MDL Rounded ML
0.30 mg/L NO3- N 0.05 0.20

Table 3. Matrix Spike (MS) / Matrix Spike Duplicate (MSD) QC Acceptance Criteria
Lower Limit
MS/MSD Spike Maximum RPD Upper Limit of
of Recovery
Concentration (%) Recovery (%)
(%)
5.0 mg/L NO3- N 20 90.0 110

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18.0 Glossary of Definitions and Purposes
The definitions and purposes are specified to this method but have been conformed to common
usage as much as possible.

18.1 Units of weight and measure and their abbreviations

18.1.1 Symbols
o
C: degrees Celsius

18.1.2 Alphabetical characters

mg/L: milligram per liter

18.2 Definitions, acronyms, and abbreviations

18.2.1 LRB Laboratory Reagent Blank

18.2.2 MDL: Method detection limit

18.2.3 ML: Minimum level of quantitation

18.2.4 IPR: Initial precision and recovery

18.2.5 OPR: On-going precision and recovery

18.2.6 MS: Matrix spike

18.2.7 MSD: Matrix spike duplicate